Laboratory Reagent Preparation Guide
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derejeLaboratory Reagent Preparation Guide
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derejePREPARING
REAGENTS
Preparation of Saline
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Preparation of Saline
Purpose
Saline is a solution that used for
different lab activity b/c of it’s isotonic
solution property.
We use saline in our laboratory
for – wet mount preparation
- cell suspension preparation
-cell washing purpose
-dilution of sample etc….
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Necessary Material
Balance
Beaker
Measuring cylinder
Nacl/table salt
Distilled water
Bottle plastic/glass
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Procedures
Measure about 500ml of distilled
water in to beaker w/h have
capacity of 1 litter/above.
Measure 8.5gm of Nacl salt on the
balance.
Pour slowly into the measured
water then mix well until complete
dissolve.
Then measure the remain 500ml
of distilled water into the beaker.
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Procedures cont.…
Mix well then transfer to bottle
NB carefully measure the salt b/c
salt amount can affect isotonic
property of saline.
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Preparation of
0.5%bleach
Purpose
Bleach solution is used in our
laboratory for disinfection
purpose and sputum
concentration techniques by
making different concentration.
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Necessary Material
Measuring cylinder
Beaker
Stock bleach solution(5%)
Bottle for use
Water
Labeling devices
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Procedures
Calculate how to prepare desired
percent of bleach by using the following
formula
C1V1=C2V2
Measure calculated volume of stock
bleach solution then pour into
measured amount of water then mix
well.
OR THE NEXT SLIDE……
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Calculate the following
1. prepare 50ml of 0.5%sodium
hypo chloride (bleach) solution.
2. prepare 60ml of 1%%sodium
hypo chloride (bleach) solution.
3. If you dilute 10ml of
concentrated bleach with 90ml of
water how much is it
concentration of the solution?
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Alcohol (Ethanol), 95% v/v
To make 100 ml:
Ethanol, absolute............................. 95 ml
Distilled water .................................... 5
ml
1 Using a 100 ml cylinder, measure 95 ml
of absolute ethanol.
2. add 5ml of distilled water mix well and
labeled with all necessary information.
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Preparation of 70% alcohol Purpose
70% alcohol is one of the anti
septic solution use in medical lab
Preparation of 70% alcohol is the
same with that of bleach
preparation
The concentrated alcohol
concentration is about 95-96% we
can use one the two.
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[Link] much ml of concentrated
alcohol is required to prepare 70ml
of 70%alcohol ?
2. Prepare 30ml of 70%alcohol
solution
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EDTA anticoagulant
EDTA is an anticoagulant that used for
preventing blood from coagulate
The most routinely used type and best
for most hematological tests. That
prevents clot by chelating calcium.
Obtain ready-prepared anticoagulated
bottles, or prepare as follows:
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Cont.…
The component of EDTA
Di-potassium ethylene- diamine-
tetra-acetic
acid ............................... 2.5 g
Distilled water......................... 25
ml
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procedure
1. Weigh the chemical, and transfer it to a small
glass bottle.
2. Measure 25 ml of water, add to the chemical,
and mix to dissolve. Label the bottle.
3. For use, pipette 0.04 ml of the reagent into
small bottles marked to hold 2.5 ml of blood.
4. Place the small bottles without tops, on a
warm bench for the anticoagulant to dry. Protect
from dust and flies.
5. When dry, replace the bottle tops, and store
ready for use.
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Sodium citrate anticoagulant
Sodium citrate anticoagulant to prevent
blood from clot like that of EDTA but we
use citrate for ESR and Coagulation test
When use for ESR test 1part citrate 4parts
of blood and for coagulation test use
1part citrate 9part blood
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procedure
To make about 100 ml:
tri-Sodium citrate ........................................... 3.8 g
Distilled water ............................................. 100 ml
1 Weigh the sodium citrate and transfer it to a
leak-proof bottle.
2 Add 100 ml of water and mix to dissolve the
chemical.
3 Label the bottle and store preferably at 2–8 °C.
Renew every 6 weeks or sooner if it becomes
contaminated (cloudy).
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Giemsa stain
Giemsa stain is used for
＊ Batch staining of large numbers of
thick and thin blood films for research
purpose.
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procedure
Purchase ready-made or prepare using
the following formula.
To make about 500 ml:
Giemsa powder .................................
3.8 g
Glycerol (glycerin)...........................
250 ml
Methanol (methyl alcohol)................
250 ml
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Procedure cont.…
1. Weigh the Giemsa on a piece of clean paper
(pre weighed), and transfer to a dry brown bottle
of 500 ml capacity which contains a few glass
beads.
Note: Giemsa stain will be spoilt if water enters the
stock solution during its preparation or storage.
2. Using a dry cylinder, measure the methanol,
and add to the stain. Mix well.
Caution: Methanol is toxic and highly flammable,
therefore handle it with care and use well away
from an open flame.
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Procedure cont.…
3. Using the same cylinder, measure the
glycerol, and add to the stain. Mix well.
4. Place the bottle of stain in a water bath at 50–
60 °C, or if not available at 37 °C, for up to 2
hours to help the stain to dissolve. Mix well at
intervals.
5. Label the bottle, and mark it Flammable and
Toxic. Store at room temperature in the dark. If
kept well-stoppered, the stain is stable for
several months. For use: Filter a small amount of
the stain into a dry stain dispensing container.
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Working solution of Giemsa
preparation
Their is three types of Giemsa
working solution are available
1. 3% Working s/on stain for
30mint
2. 5% Working s/on stain for
15mint
3. 10% Working s/on stain for
10mint
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Procedure for preparing working
s/on
Calculating the sock solution as
100%concentrated the by using
the previous formula we can
calculate to prepare as you need.
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Calculate the following
Prepare 50ml of 3%,5% and 10%
of Giemsa working solutions.
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Buffered saline, pH 7.0–7.1
This can be prepared by adding 0.4–0.7
ml of 10 g/l di-sodium hydrogen
phosphate* to every 500 ml of
physiological saline. Check the pH using
narrow range pH papers or a pH meter.
* Prepare by dissolving 1 g di-sodium
hydrogen phosphate in 100 ml distilled
water.
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Formol water, 10% v/v
Formol water 10% is used for
preservative of stool and urine sample
and for stool concentration techniques
The concentrated formal dehyde
solution have the concentration of 37-
41%
We can prepare 5% Formol water
solution for preserving trophozoite
stage of parasite
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Procedure
1 Measure the water and transfer it to a leak-
proof bottle.
2 Measure the formaldehyde solution and add to
the water. Mix well.
Caution: Concentrated formaldehyde solution is
a toxic chemical with a vapour that is irritating to
the eyes and mucous membranes, therefore
handle it with great care in a well-ventilated
room.
3 Label the bottle and store at room temperature
in a safe place. The reagent is stable indefinitely.
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Prepare 100ml of 10% and 5%
formol saline solution.
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Iodine (Dobell’s) for faecal
preparations
Iodine is solution used for preparation of
wet smear for differentiating larval stage
of parasite.
To make about 100 ml:
Potassium iodide........................ 4 g
Iodine........................................ 2 g
Distilled water ........................... 100 ml
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procedure
1. Weigh the potassium iodide and dissolve completely in
about 50 ml of the water.
2. Weigh the iodine and add to the potassium iodide
solution. Mix well to dissolve.
Caution: Iodine is injurious to health if inhaled or allowed to
come in contact with the eyes, therefore handle with care in
a well-ventilated room.
3. Add the remainder of the water, and mix. Transfer to a
brown bottle.
4. Label the bottle, and mark it Harmful. Store in the dark at
room temperature. The reagent is stable for several months.
For use: Transfer to a small brown bottle with a cap into
which a dropper can be inserted.
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Zinc sulphate solution, 33% w/v
Zinc sulphate solution is used in
floatation techniques of stool
concentration
To make approximately 500 ml:
Zinc sulphate .................................
165 g
Distilled water ................................
500 ml
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Procedure
1. Weigh the zinc sulphate and transfer it to
a leak-proof bottle of about 1 litter capacity.
2. Measure the water and add it to the
chemical.
3. Stopper the bottle, and mix well.
4. Stand the bottle in a container of hot
water to dissolve the zinc sulphate. Mix until
the chemical is completely dissolved.
5. Allow the solution to cool to room
temperature
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Procedure cont.
6. Using a hydrometer, check the relative
density of the solution. If the density is not
within 1.180–1.200, add more chemical or
water to bring the solution within the correct
density range.
7. Label the bottle and store at room
temperature. When stored with the bottle top
tightly stoppered, the solution is stable for
several
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Quiz 2
1. prepare 45ml of normal saline solutions
2. calculate to prepare 150ml of Giemsa stock
solution.
3. prepare 5%Giemsa working solution of 100ml
4. prepare 110ml of 1%bleach solution for bench
cleaning.
5. prepare 50ml of skin antiseptic solution for
sample collection work station.
6. To prepare 40ml of 5% formalin solution how
much volume(ml) of stock is needed?
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